The present invention relates to producing a spider silk protein, protein fragment or variant, in commercially useful amounts, by means of a recombinant host organism.
There is considerable interest currently in making high-strength, light and versatile fibers. Most high-strength fibers used today, such as Nylon.TM. and the synthetic ballistic fiber Kevlar.TM., have a high density, are expensive and are limited in their range of use. If it were available in commercially useful amounts, a naturally occurring protein could provide an alternative fiber with enhanced properties.
Spider silks have been demonstrated to have several desirable characteristics. For example, spider silk could be used as a light-weight, high-strength fiber for various textile and ballistics applications, as well as for composite materials. Spider silks represent a very diverse group of fibers, particularly with respect to their mechanoelastic properties, which in turn are largely a function of fiber composition and molecular conformation. The spider silks range from those displaying a tensile strength greater than steel (7.8 vs 3.4 G/denier) and those with an elasticity greater than wool (46% vs 43% extension to break) to others characterized by energy-to-break limits that are greater than Kevlar.TM. (1.times.10.sup.5 vs 3.times.10.sup.4 JKG-1).
Considerable difficulty has been encountered in attempting to solubilize and purify natural spider silk while retaining the molecular-weight integrity of the fiber. Another disadvantage of spider silk protein is that only small amounts are available from cultivated spiders, making commercially useful quantities of silk protein unattainable at a reasonable cost.
The term "fibroin" is often used for the silk fibers secreted by some insects and arachnids. See, e.g., Lucas et al., Adv. Protein Chem. 13:107-242 (1958). Studies of the chemistry of these fibroins have been reported, for example, by Work and Young, J. Arachnol. 15:65-80 (1987). Nevertheless, only limited data are available on the composition of silk fiber from spiders, including those of the genus Nephila. For example, partial amino-acid constituency has been reported for silks of N. senegalensis and N. madagascar lensis, Lucas et al., J. Mol. Biol. 2:339-49 (1960); and N. clavipes, Tillinghast & Christensen, J. Arachnol. 10: 69-74 (1984). While these investigations suggest that the different Nephila silks vary in composition and properties, there is insufficient information to make a definitive correlation between chemical composition and structural properties.
The silk fibers of Nephila spiders are synthesized by specialized glands situated in the abdominal cavity. Andersen has reported on the amino acid compositions for the seven silks obtained from one animal. See Andersen, Como. Biochem. Physiol. 35:705-711 (1970). Of the seven types of silks, only two have been investigated in any detail, and no sequence data were obtained.
In addition to the problem of solubility, multiple forms of spider silks are produced simultaneously by any given spider. The resulting mixture is much less useful than a single isolated silk because the different spider-silk proteins have different properties and, due to solubilization problems, are not easily separated by methods based on their physical characteristics.
Accordingly, at least three major problems have inhibited consideration of the feasibility of producing spider silk fibers with desirable characteristics, in commercially useful quantities, for use as components of textile, composite and ballistic materials. First, an effective means for solubilizing silk protein has been unavailable heretofore, making amino-acid sequencing of the protein virtually impossible. As a corollary, it has not been possible to identify, isolate and clone a spider silk-encoding DNA into a suitable expression system. Nor has it been feasible to produce a spider silk protein in quantities much greater than can be obtained naturally from spiders. Consequently, there has been a need but no means for providing commercially useful quantities of spider silk protein in a form displaying homogeneous mechanoelastic properties.